| Grass carp is one of the main species of freshwater aquaculture in China and has important economic value.However,grass carp hemorrhagic disease caused by grass carp reovirus（GCRV）has a very high mortality rate,causing huge economic losses to the grass carp breeding industry.The GCRV genome consists of 11 segmented double-stranded RNA fragments,encoding 11-12 proteins,of which the outer capsid protein of the virus is often used as an antigenic protein for subunit vaccines due to its good immunogenicity.In this dissertation,we take GCRV106 as the research object,and the subunit vaccines of the viral coat proteins VP35 and VP4 were prepared based on the baculovirus expression system,and the immune protection effects of the vaccine were evaluated by injection and oral administration.The main findings are as follows:1.Study on the expression of recombinant protein and its immunoprotective effect on rare salamander（1）Using the GCRV 106 virus genome as a template,the VP35,VP4 and VP35-VP4genes were obtained by PCR amplification and inserted into the baculovirus transfer vector p Fast Bac TMⅠto construct donor plasmids p Fast Bac-VP35,p Fast Bac-VP4 and p Fast Bac-VP35-VP4.SDS-PAGE and Western blot results showed that the fusion proteins VP35 and VP4 of about 34 k Da and 66 k Da were expressed after infection with SF9 cells.After purification of the recombinant proteins VP35,VP4 and VP35-VP4 by Ni column affinity chromatography,the concentrations determined by BCA were 0.3 mg/m L,0.2 mg/m L and0.2 mg/m L,respectively.The immunization experiment was divided into three immunization groups,and immune rare minnow was injected intraperitoneally at 3μg per tail.（2）The expression of immune-related genes was detected by q PCR.The results showed that the expression levels of Ig M,TNF-α,My D88,NF-Kb,IL-1β,Mx and TLR3 in the immunized group VP35,VP4 and VP35-VP4 were all present in the liver,spleen and kidney at different time points after immunization It was up-regulated in different degrees,and the change trend of the three immunization groups was the same.Compared with the control group,the expression levels of IL-1βand TLR3 in the spleen and kidney were significantly up-regulated and reached a peak at the 3 dpi,up-regulated by 2-6 fold（P<0.01）.The relative expression of Mx in the liver and spleen was up-regulated at the 3 dpi and reached the highest value at the 5 dpi,up-regulated by 3-6 fold（P<0.01）.NF-k B and TNF-αpeaked in the liver,spleen and kidney at the 14dpi,and were increased 4-9 fold（P<0.01）.Ig M was up-regulated at the 7 dpi and reached its peak at the 21 dpi.It was up-regulated 8-20 fold in the liver,spleen and kidney（P<0.01）.（3）The challenge was performed 21 d after immunization.The results showed that the recombinant proteins r VP35,r VP4 and r VP35-VP4 can protect the rare minnow from GCRV infection to varying degrees,and the immune protection rates were 33%,60%,and67%,of which the r VP35-VP4 protein has the best immune protection effect.2 Preparation of oral vaccine and study on its immune protection effect on grass carp（1）The Bac-to-Bac silkworm baculovirus expression system was used to express the recombinant protein r VP35-VP4 in Bm E cells and 5th-instar silkworm,and a large amount of green fluorescence was observed in infected Bm E cells through IFA.5th-instar silkworms were collected 120 h after infection with r Bm Bac-VP35-VP4,and freeze-dried under vacuum to form a lyophilized powder,which was added to the feed at 5%to orally immunized grass carp.（2）The results of red and white blood cell count showed that at 14,21,and 28 days after immunization,the amount of WBC in grass carp in the immunized group increased by 1.4-1.7 fold（P<0.05）compared with the control group.WBC differential counting results showed that at the 14 dpi,the percentage of neutrophils and monocytes in leukocytes increased and was significantly higher than the control group（19.82±1.18%,10.12±1.18%）（P<0.05）,and it decreased at the 28 dpi,but the percentage of lymphocytes in leukocytes increased significantly（93.14±0.82%）（P<0.05）.The ELISA test results showed that the titer of Ig M antibody in the immunized group increased significantly two weeks after the immunization,and reached the highest level at the 28 dpi,with an OD450of 0.71±0.04（P<0.01）.The results of q PCR showed that the expression levels of immune-related genes IFN I,TLR22,IL-1β,MHC I,Mx,and Ig M were significantly upregulated at different time points after oral administration of grass carp.Compared with the control group,IFN I in the liver and spleen began to increase and reached a peak at the21 dpi,respectively 2.2 fold（p<0.05）and 2.8 fold（p<0.01）.IL-1βand MHCⅠin the spleen began to increase and reached the peak at the 21 dpi,respectively,2.8 fold（P<0.01）and 2.2 fold（P<0.05）.The Ig M in the liver and hindgut started to increase at the 14 dpi,and reached the highest value at the 21 dpi,which was increased by 3.1 fold（P<0.01）and6.5 fold（P<0.01）,and in the kidney and spleen,at the 14 dpi.It starts to increase at the 21dpi and reaches the peak at the 28 dpi,and increases 9 fold（p<0.01）and 6.8 fold（p<0.01）.（3）The results of the challenge test showed that the immune protection rate of grass carp for oral silkworm lyophilized powder vaccine was 56%,which was significantly higher than the control group（P<0.01）.The above results show that the oral silkworm lyophilized powder vaccine can induce grass carp to produce an immune response and effectively protect grass carp from GCRV infection.This study lays the foundation for the development of GCRV oral subunit vaccine. |
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